Most solid tumors and their metastases experience regions of hypoxia, which promotes both tumor progression and resistance to therapy. Hypoxia is also important for the proliferation and maintenance of cancer stem cells (CSC), a minority population within the heterogenous tumor mass capable of generating the diverse tumor cell population and implicated in tumor recurrence following therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1 and HIF-2, non-redundant transcription factors regulating both overlapping and unique downstream target genes. HIF-2 is an important driver of tumor morbidity in renal cell carcinoma (RCC), neuroblastoma, glioblastoma (GBM) and non small cell lung cancer (NSCLC). This has been linked with the ability of HIF-2 (but not HIF-1) to drive proliferation, invasion and to promote the maintenance and growth of cancer stem cells within these tumor types. In this setting, high HIF-1 predicts for better patient prognosis. Hence, we propose that the specific inhibition of HIF-2 will be a useful strategy for treating HIF-2 driven tumors and for targeting the CSC population. Our goal is to identify small molecule specific inhibitors of HIF-2 that will lead to the development of ne anti-cancer therapies. We have generated 786-0 RCC cells stably expressing a hypoxia responsive element (HRE)-luciferase construct. The 786-0 HRE cells do not express HIF-1 and we will use these cells to screen the NIH MLSMSR collection for inhibitors of HIF-2. We have performed the necessary optimization assays for the 786-0 HRE cells in 384-well plates and find that these cells are suitable for HTS using our pan-HIF inhibitor PX478 as a positive control. Signal to background ratios were ~20, Z' factor for optimized assay conditions was 0.67, maximum DMSO concentration tolerated was 0.75% (v/v) and total assay time from cell seeding to luciferase reading was 48 hours. As a counter screen, we have generated the MIAPACA-2 HRE cells that only express HIF-1 and we will use these cells to eliminate hits that also inhibit HIF-1. As a validation screen, we will use the PANC HRE cells in which HRE luciferase activity is HIF-2 dependent to confirm HIF-2 inhibition by hits from the primary screen. As an indicator of potential anti-tumor activity, hit compounds will be evaluated using secondary assays to determine their ability to inhibit HIF-2 downstream target genes and functional outcomes such as epithelial to meseenchymal transition and 3D colony formation in relevant cell models such has RCC and GBM. The proposed screen will lead to the identification of novel HIF-2 specific inhibitors that will provide structural leads for new anti-cancer therapies. Additionally, this screen will also facilitate the identification pan HIF-1/ HIF-2 inhibitor that may have therapeutic application and provide new insights into novel mechanisms of HIF-1/2 is form specific regulation.